Cephalosporin compounds, and antibacterial agents

ABSTRACT

A cephalosporin compound having the formula: ##STR1## wherein R 1  is a hydrogen atom, a halogen atom, a methoxy group, a substituted or unsubstituted vinyl group, or --CH 2  --A wherein A is a hydrogen atom, an azido group, an acyloxy group, a carbamoyloxy group, a subsituted or unsubstituted heterocyclic group (wherein the hetrocyclic ring is a 5- or 6-membered heterocyclic ring having from 1 to 4 oxygen, nitrogen or sulfur atoms), or a substituted or unsubstituted heterocyclic thio group (wherein the heterocyclic ring is a monocyclic or bicyclic heterocyclic ring having from 1 to 5 oxygen, nitrogen or sulfur atoms), or a pharmaceutically acceptable salt thereof.

The present invention relates to novel cephalosporin compounds,processes for their preparation, antibacterial agents containing themand novel intermediate compounds for the preparation thereof.

A number of cephalosporin antibiotics including cefazolin and cephalotinare known. However, none of cephalosporin antibiotics are fullysatisfactory with respect to the antibacterial activities particularlyagainst Pseudomonas aeruginosa among gram-negative bacteria.

The present inventors have found that by the introduction of a novelsubstituent at the 7-position of the cephem ring, it is possible toobtain a cephalosporin compound having remarkable antibacterialactivities against both gram-positive bacteria and gram-negativebacteria, particularly against Pseudomonas aeruginosa. The presentinvention is based on such a discovery.

The present invention provides a cephalosporin compound having theformula: ##STR2## wherein R₁ is a hydrogen atom, a halogen atom, amethoxy group, a substituted or unsubstituted vinyl group, or --CH₂ --Awherein A is a hydrogen atom, an azido group, an acyloxy group, acarbamoyloxy group, a substituted or unsubstituted heterocyclic group(wherein the heterocyclic ring is a 5- or 6-membered heterocyclic ringhaving from 1 to 4 oxygen, nitrogen or sulfur atoms), or a substitutedor unsubstituted heterocyclic thio group (wherein the heterocyclic ringis a monocyclic or bicyclic heterocyclic ring having from 1 to 5 oxygen,nitrogen or sulfur atoms), or a pharmaceutically acceptable saltthereof.

Now, the present invention will be described in detail with reference tothe preferred embodiments.

The heterocyclic group for A includes a substituted or unsubstitutedtetrazolyl group. Likewise, the heterocyclic thio group for A may be asubstituted or unsubstituted thiazolylthio, isothiazolylthio,thiadiazolylthio, triazolylthio, triazinylthio, tetrazolylthio,triazolopyrimidinylthio, 1-substituted pyridinium-4-ylthio or2,3-cyclopenteno-1-substituted pyridinium-4-ylthio group.

The thiadiazolylthio group may be, for example, a 1,3,4-thiadiazolylthiogroup, a 1,2,3-thiadiazolylthio group, or a 1,2,4-thiadiazolylthiogroup. The triazolylthio group may be, for example, a1H-1,2,3-triazolylthio group, a 4H-1,3,4-triazolylthio group, or a2H-1,2,4-triazolylthio group. Likewise, the triazolopyrimidinylthiogroup may be, for example, an s-triazolo[1,5-a]pyrimidinylthio group, ora 1H-triazolo[4,5-e]pyrimidinylthio group.

Further, the substituted on the heterocyclic rings may be, for example,a lower alkyl group, a carboxymethyl group, a lower alkoxycarbonylmethylgroup, a hydroxy-(lower)alkyl group, a di-(lower)alkylamino-(lower)alkylgroup, a carboxyl group, a hydroxyl group, an oxo group, or an aminogroup.

Now, specific examples of the compound of the present invention will begiven, but it should be understood that the present invention is notlimited to such specific compounds.

(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3(1,2,3-thiadiazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3(2-carboxy-5-methyl-s-triazolo[1,5-a]pyrimidin-7-ylthiomethyl)-3-cephem-4-carboxylicacid.

(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3(1-methyl-1H-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3[1-(2-hydroxyethyl)-1H-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3(1-carboxymethyl-1H-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-[1-(2-dimethylaminoethyl)-1H-tetrazol-5-ylthiomethyl]-3-cepham-4-carboxylic acid.

(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(1,3,4-thiadiazol-5-ylthiomethyl)-3-cephem-4carboxylicacid.

(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(2-methyl-1,3,4-thiadiazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

(6R, 7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon(1,2,3-triazol-5-ylthiomethyl)-3-cephem-4carboxylic acid.

(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-as-triazin-3-ylthiomethyl)-3-cephem-4-carboxylicacid.

(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3(5-ethoxycarbonylmethyl-4-methylthiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid.

(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(4-carboxy-3-hydroxyisothiazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3(acetoxymethy-3-cephem-4-carboxylic acid.

(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-vinyl-3-cephem-4-carboxylicacid.

(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-[(5-methyl-2H-tetrazol-2yl)methyl]-3-cephem-4-carboxylicacid.

(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(5-carboxymethyl-4-methylthiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid.

(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(1-methylpyridinium-4-ylthiomethyl)-3-cephem-4-carboxylate.

(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(2,3-cyclopenteno-1-methylpyridinium-4-ylthiomethyl)-3-cephem-4-carboxylate.

Sodium salt of (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(1-carboxymethylpyridinium-4-ylthiomethyl)-3-cephem-4-carboxylicacid.

Sodium salt of (6R,7R)-7-[(Z)-2-(2-aminothiazol-4yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(1-carboxymethyl-2,3-cyclopentenopyridinium-4-ylthiomethyl)-3-cephem-4-carboxylic acid.

(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3(1-t-butoxycarbonylmethylpyridinium-4-ylthiomethyl)-3-cephem-4-carboxylate.

(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3(1-t-butoxycarbonylmethyl-2,3-cyclopentenopyridinium-4-ylthiomethyl)-3-cephem-4-carboxylate.

(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(5-metyl-s-triazolo[1,5-a]pyrimidin-7-ylthiomethyl)-3-cephem-4-carboxylicacid.

The compounds of the formula I of the present invention may be used asthey are or in the form of their salts. The salts are pharmaceuticallyacceptable nontoxic salts with an acid or with a base. The salts with anacid include salts with an inorganic acid such as a hydrogen halide acid(e.g. hydrochloric acid or hydrobromic acid) or sulfuric acid, and saltswith an organic acid such as fumalic acid or citric acid. The salts witha base include an alkali metal salt such as a sodium salt or a potassiumsalt, and salts with an organic base such as an ammonium salt, adicyclohexylamine salt, a triethylamine salt, an ethanolamine salt, anornithine salt or a lysine salt. Further, the compounds of the formula Iof the present invention have (Z)-isomer and (E)-isomer because of thepartial structure represented by the formula: ##STR3## Such isomers andthird mixtures are included in the scope of the present invention.

Furthermore, the compounds of the formula I of the present inventionhave the following keto and enol tautomeric isomers because of thepartial structure represented by the formula: ##STR4## Such isomers andtheir mixtures are included in the scope of the present invention. Forthe convenience sake, however, the compounds will be named by theketo-form.

The compounds of the formula I of the present invention may be preparedby various processes. Typical processes will be described below.

Process 1

The compound of the formula I of the present invention can be preparedby reacting a compound having the formula: ##STR5## wherein R2 is ahydrogen atom or an amino-protecting group, and each of R₃ and R₄ is ahydrogen atom or a hydroxyl-protecting group, or a reactive derivativethereof, with a compound having the formula: ##STR6## wherein R₁ is asdefined above, and R₅ is a hydrogen atom or a carboxyl-protecting group,to form a compound having the formula: ##STR7## wherein R₁, R₂, R₃, R₄and R₅ are as defined above, and, if necessary, removing theamino-protecting group, the hydroxyl-protecting group and thecarboxyl-protecting group in the compound of the formula IV.

The amino-protecting group for R₂ includes a tri-lower alkylsilyl groupsuch as a trimethylsilyl group, an acyl group such as a formyl group, achloroacetyl group, a p-methoxybenzyloxycarbonyl group, at-butoxycarbonyl group, a 2,2,2-trichloroethoxycarbonyl group, or ap-nitrobenzyloxycarbonyl group, and an aralkyl group such as a benzylgroup, a p-methoxybenzyl group, a p-nitrobenzyl group, a benzhydrylgroup (a diphenylmethyl group) or a trityl group (a triphenylmethylgroup). The hydroxyl-protecting group for R₃ and R₄ includes a tri-loweralkylsilyl group such as a trimethylsilyl group, an acyl group such as aformyl group, an acetyl group, a propionyl group, a methoxyacetyl group,or a methoxypropionyl group, an aralkyl group such as a benzyl group, ap-methoxybenzyl group, a p-nitrobenzyl group, a benzhydryl group, or atrityl group, a methoxymethyl group, an allyl group, and a pyranylgroup. The carboxyl-protecting group for R₅ includes a tri-loweralkylsilyl group such as a trimethylsilyl group, a benzhydryl group, aβ-methylsulfonylethyl group, a phenacyl group, a p-methoxybenzyl group,a t-butyl group, a p-nitrobenzyl group, and a 2,2,2-trichloroethylgroup.

In the above process, each reaction is conducted usually in a solvent ata reaction temperature of from -50° to 50° C. There is no particularrestriction as to the solvent so long as it is inert to the reaction.Preferably, however, acetonitrile, dimethyl sulfoxide, dichloromethane,dichloroethane, chloroform, ethyl ether, ethanol, dioxane,tetrahydrofuran, acetone, ethyl acetate, dimethylformamide ordimethylacetamido is used. These solvents may be used alone or incombination as a proper mixture. Further, different solvents may beemployed for the respective reaction stages.

The compound of the formula II may be used in the form of a freecarboxylic acid, or may be used for the reaction in the form of a saltor in the form of a reactive derivative of the carboxylic acid. Asuitable reactive derivative includes an acid halide (such as an acidchloride or an acid bromide), an active ester (such as a benzotriazoleester, a cyanomethyl ester, a nitrophenyl ester, an N-hydroxysuccinimideester or an N-hydroxyphthalimide ester), a mixture of acid anhydrides(such as a mixture of acid anhydrides with ethoxycarboxylic acid,isobutoxycarboxylic acid and trimethyl acetate), an active amide, and anactive azide. When the compound of the formula II is used in the form ofa free carboxylic acid, it is preferred to employ a condensation agentsuch as N,N'-dicyclohexylcarbodiimide or N,N'-diethylcarbodiimide.Depending upon the type of the reactive derivative of the carboxylicacid to be used, it may be suitable in some cases to conduct thereaction in the presence of a base in order to carry out the reactionsmoothly. As a base to be used in such a case, there may be mentioned aninorganic base such as sodium hydrogen carbonate or potassium carbonate,or an organic base such as trimethylamine, triethylamine,dimethylaniline, pyridine, N-methylmorpholine, dicyclohexylamine ordiethylamine. The removal of the protective groups may be conducted bycommon methods based on the properties of the protective groups for theamino, hydroxyl or carboxyl groups to remove the amino protecting groupR₂, hydroxyl-protecting groups R₃ and R₄ and carboxyl-protecting groupR₅ in the formula IV.

Process 2

Among the compounds of the present invention, those represented by theformula: ##STR8## wherein Het is a substituted or unsubstitutedheterocyclic group (wherein the heterocyclic ring is a monocyclic orbicyclic heterocyclic ring having from 1 to 5 oxygen, nitrogen or sulfuratoms), can be prepared by reacting a compound having the formula:##STR9## wherein R₂ is a hydrogen atom or an amino-protecting group, andeach of R₃ and R₄ is a hydrogen atom or a hydroxyl-protecting group,with a compound having the formula: ##STR10## wherein R₅ is a hydrogenatom or a carboxyl-protecting group, and X is a chlorine atom, a bromineatom, an iodine atom or an acetoxy group, to form a compound having theformula: ##STR11## wherein R₂, R₃, R₄, R₅ and X are as defined above,and then reacting the compound of the formula VI with a compound havingthe formula:

    HS--Het                                                    (VII)

wherein Het is as defined above, to form a compound having the formula:##STR12## wherein R₂, R₃, R₄, R₅ and Het are as defined above, and, ifnecessary, removing the amino-protecting group, the hydroxyl-protectinggroup and the carboxyl-protecting group in the compound of the formulaVIII.

In the above process, each reaction is conducted usually in a solvent ata reaction temperature of from -50° to 50° C. There is no particularrestriction as to a solvent so long as it is inert to the respectivereactions. Preferably, however, acetonitrile, dimethylsulfoxide,dichloromethane, dichloroethane, chloroform, ethyl ether, methanol,dioxane, tetrahydrofuran, acetone, ethyl acetate, dimethylformamide ordimethylacetamide is used. These solvents may be used alone or in acombination as a proper mixture. Further, different solvents may be usedfor the respective reaction stages.

The compound of the formula VII may be used in the free form, but it mayadvantageously be used in the form of an alkali metal salt such as asodium salt or a potassium salt. The reaction time varies depending uponthe starting material, solvent, etc., and is usually suitably selectedwithin a range of a few hours to a few days. The reaction is conductedusually at a pH of from 2 to 8, preferably at a neutral level. In somecases, a quaternary ammonium salt having a surface active function suchas trimethylbenzylammonium bromide or triethylbenzylammonium hydroxide,may be added to the reaction system to conduct the reaction smoothly.Further, in order to prevent the oxidation in air, the reaction may beconducted in an inert atmosphere such as nitrogen, whereby anadvantageous results will be obtained.

The removal of the protective groups may be conducted by common methodsbased on the properties of the protective groups for the amino, hydroxylor carboxyl groups, to remove the amino-protecting group R₂,hydroxyl-protecting groups R₃ and R₄ and carboxyl-protecting group R₅ inthe formula VIII.

As shown in Tables 1 and 2 given hereinafter, compounds of the formula Iof the present invention exhibit excellent anti-bacterial activities andinfection-preventive effects. They show excellent activities againstsome pathogenic bacteria belonging to gram-positive bacteria. Aparticularly important feature is that they show remarkableeffectiveness against Pseudomonas aeruginosa. Thus, the compounds of theformula I and their salts according to the present invention are usefulas anti-bacterial agents or chemical treatment agents for warm bloodanimals including human beings and domestic animals. They are useful forcuring infectious diseases caused by gram-positive bacteria andgram-negative bacteria. They are also useful as additives to animalfeeds.

The compounds of the formula I and their salts according to the presentinvention may be administered orally or non-orally (e.g. intravenousadministration, intramuscular administration or hypodermicadministration). The dose varies depending upon the age, weight,condition and the degree of disease of the patient. Usually, however,the dose per day is from about 0.2 to about 10 g, preferably from 0.5 to4.0 g. The compound of the formula I or its salt may be used also as apharmaceutical drug having a pharmaceutically acceptable vehicleincorporated to be suitable for oral or non-oral administration. Thepharmaceutically acceptable vehicle includes gelatin, lactose, fructose,sodium chloride, starch, magnesium stearate, talc, vegetable oil andother pharmaceutical vehicles. The pharmaceutical drug may be a solidformulation such as tablets, granules, capsules, microcapsules orpowder, or may be a liquid formulation such as a solution, a suspensionor an emulsion. Further, if necessary, an adjuvant, a stabilizer, awetting agent, an emulsifier or other conventional additives may beincorporated.

Another object of the present invention is to provide an intermediatecompound of the formula II useful for the preparation of the compound ofthe formula I of the present invention.

The intermediate compound of the formula II includes a compound havingthe formula: ##STR13## wherein R₂, R₃ and R₄ are as defined above, orits reactive derivative. As a suitable reactive derivative, there may bementioned, for example, an acid halide (such as an acid chloride or anacid bromide), an active ester (such as benzotriazole ester, cyanomethylester, nitrophenyl ester, N-hydroxysuccinimide ester orN-hydroxyphthalimide ester), a mixture of acid anhydrides (such as amixture of acid anhydrides comprising ethoxycarboxylic acid,isobutoxycarboxylic acid and trimethyl acetate), an active amide, and anactive azide.

A typical method for the preparation of the intermediate compound of theformula II of the present invention will be described. A compound of theformula X wherein R₃ is as defined above, is obtained by protecting thephenolic hydroxyl group of kojic acid of the formula IX by aconventional method. This compound of the formula X is treated in awater-containing alcohol with hydroxylamine hydrochloride and sodiumacetate at a temperature of from room temperature to 60° C. to obtain acompound of the formula XI. A compound of the formula XII wherein R₄ isas defined above is obtained by protecting the N-oxide of the compoundof the formula XI by a conventional method. The compound of the formulaXII is reacted with N-hydroxyphthalimide in an anhydrous solvent (suchas dioxane, tetrahydrofuran, dimethylformamide or dimethylacetamide) inthe presence of triphenylphosphine and azodicarboxylic acid diethylester, to obtain a compound of the formula XIII. This compound of theformula XIII is treated with hydrazine to obtain a compound of theformula XIV, which is then reacted with a compound of the formula XV, toobtain an intermediate compound of the formula II.

Now, the present invention will be described in further detail withreference to Test Examples, Reference Examples and working Examples.However, it should be understood that the present invention is by nomeans restricted to such specific Examples.

TEST EXAMPLE 1: Measurement of minimum inhibitory concentration

The minimum inhibitory concentration (M.I.C.) (μg/ml) of each testcompound was measured by an agar plate dilution method according to thestandard method by Japan Chemical Therapy Academy by using aMuller-Hinton agar culture medium (manufactured by Nissui K.K.). As thetest bacteria, Staphylococus aureus FDA 209-P JC-1, Escherichia coli ML4707, Klebsiella pneumoniae No. 42, Proteus vulgaris No. 33, Serratiamarcescens No. 16-2, Enterobacter cloacae Nek 39, Acinetobactercalcoaceticus No. 4, Pseudomonas aeruginosa K-13, Pseudomonas aeruginosaY-1 and Pseudomonas cepacia 23 were employed. The results are shown inTable 1.

                                      TABLE 1                                     __________________________________________________________________________    (μg/ml)                                                                                 Compound (Example No.)                                           Bacteria     3  4  5   6   18  19 CTX CAZ                                                                              CFS                                  __________________________________________________________________________    S. aureus FDA 209-P JC-1                                                                   6.25                                                                             12.5                                                                             50  25  6.25                                                                              12.5                                                                             3.13                                                                              12.5                                                                             3.13                                 E. coli ML 4707                                                                            0.05                                                                             0.1                                                                              ≦0.025                                                                     ≦0.025                                                                     ≦0.025                                                                     0.05                                                                             ≦0.025                                                                     0.2                                                                                50                                 K. pneumoniae No. 42                                                                       0.1                                                                              0.2                                                                              ≦0.025                                                                     0.05                                                                              0.05                                                                              0.1                                                                              0.1 0.39                                                                             >100                                 P. vulgaris No. 33                                                                         0.05                                                                             0.1                                                                              ≦0.025                                                                     0.05                                                                              0.1 0.2                                                                              0.05                                                                              0.1                                                                              --                                   S. marcescens No. 16-2                                                                     0.78                                                                             0.39                                                                             0.39                                                                              0.39                                                                              0.78                                                                              1.56                                                                             6.25                                                                              1.56                                                                             >100                                 E. cloacae Nek 39                                                                          0.78                                                                             0.39                                                                             0.39                                                                              0.78                                                                              0.39                                                                              0.2                                                                              3.13                                                                              1.56                                                                             >100                                 A. calcoaceticus No. 4                                                                     3.13                                                                             12.5                                                                             1.56                                                                              3.13                                                                              3.13                                                                              6.25                                                                             25  6.25                                                                               50                                 P. aeruginosa K-13                                                                         0.39                                                                             0.2                                                                              0.1 0.2 0.78                                                                              1.56                                                                             25  3.13                                                                             3.13                                 P. aeruginosa Y-1                                                                          0.2                                                                              0.39                                                                             0.1 0.1 0.2 0.78                                                                             12.5                                                                              1.56                                                                             1.56                                 P. cepacia 23                                                                              0.1                                                                              0.2                                                                              0.05                                                                              0.1 0.2 0.39                                                                             3.13                                                                              1.56                                                                             >100                                 __________________________________________________________________________     (Inoculated amount of cells: 10.sup.6 CFU/ml                                  CTX: cefotaxime;                                                              CAZ: ceftazidime;                                                             CFS: cefsulodin                                                          

TEST EXAMPLE 2: Infection-preventive effects

JCL:JCR type male mice of 4 weeks old having a body weight of from 17 to19 g were used in groups each consisting of from 6 to 10 animals. Astest bacteria, Staphyloccocus aureus Smith, Escherichia coli ML 4707,Pseudomonas aeruginosa K-13, and Pseudomonas aeruginosa Y-1 were used.The test bacteria were cultured at 37° C. for 16 hours by using anutrient broth. Depending upon the respective bacteria, bacterialsolutions containing from 0.5 to 5% of mucin (Pseudomonas aeruginosaK-13 and Y-1) or bacterial solutions containing no mucin (Staphyloccocusaureus Smith and Escherichia coli ML 4707) were prepared. The bacterialsolutions were intraperitoneally inoculated to mice in an amount 0.5 mleach, and upon expiration of 1 hour, solutions containing test compoundswere hypodermically administered in the respective doses. Then, the micewere observed for survival or death for 7 days. From the survival ratesduring the 7 days, ED₅₀ values were obtained by a Probit method. Theresults are shown in Table 2.

                                      TABLE 2                                     __________________________________________________________________________    ED.sub.50 (mg/kg)                                                             Bacteria   Compound (Example No.)                                             (Inoculated amount)                                                                      3  4  5  6  18  19  CTX   CAZ                                                                              CFS                                   __________________________________________________________________________    S. aureus Smith                                                                          12 8.7                                                                              -- 42 2.9 --  3.1   17 --                                    7.7 × 10.sup.7 cells/mouse                                              E. coli ML 4707                                                                          0.11                                                                             0.16                                                                             0.12                                                                             0.13                                                                             0.034                                                                             0.077                                                                             0.095 0.73                                                                             >100                                  1.1 × 10.sup.6 cells/mouse                                              P. aeruginosa K-13                                                                       9.7                                                                              12 18.9                                                                             4.6                                                                              13  9.6 >100  31 39                                    3.0 × 10.sup.4 cells/mouse                                              P. aeruginosa Y-1                                                                        2.6                                                                              4.0                                                                              -- 1.4                                                                              2.0 7.8 >200  16 13                                    4.0 × 10.sup.5 cells/mouse                                              __________________________________________________________________________

EXAMPLE 1

(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)aceticacid ##STR15##

This compound was produced by the following Steps (1) to (5).

(1) 5-benzhydryloxy-2-hydroxymethyl-4-pyrone ##STR16##

14.2 g (0.1 mol) of kojic acid was added to ethanol (400 ml), and heatedto 60° C. and dissolved. After cooling the solution to room temperature,29.1 g (0.15 mol) of diphenyldiazomethane was added thereto, and themixture was reacted at room temperature for 18 hours under stirring. Thereaction solution was concentrated to dryness, and benzene (300 ml) wasadded thereto. Then, insolubles were removed by filtration. To thefiltrate, water (300 ml) was added, and the precipitates thereby formedwere collected by filtration, and washed with benzene to obtain 17.5 g(yield: 56.8%) of the above identified compound.

NMR(CDCl₃)δ(ppm): 4.33(2H,s), 6.29(1H,s), 6.49(1H,s), 7.36(10H,s)7.44(1H,s).

(2) 1-Hydroxy-2-hydroxymethyl-5-benzhydryloxy-4-pyridone ##STR17##

17.5 g (0.0568 mol) of the product of Step (1) was added and dissolvedin a mixture of ethanol (60 ml) and water (60 ml). Then, 39.5 g (0.568mol) of hydroxylamine hydrochloride and 77.2 g (0.568 mol) of sodiumacetate trihydrate were added thereto, and the mixture was reacted at60° C. for 18 hours under stirring. Formed precipitates were collectedby filtration, washed sequentially with water, ethanol and ethyl ether,and then dried to obtain 8.1 g (yield: 44.0%) of the above identifiedcompound.

NMR(DMSO-d₆)δ(ppm): 2.71(2H,s), 6.65(1H,s), 6.87(1H,s),7.26-7.63(10H,m), 7.93(1H,s).

(3) 1,5-Dibenzhydryloxy-2-hydroxymethyl-4-pyridone ##STR18##

8.1 g (0.0251 mol) of the product of Step (2) was added todimethylsulfoxide (125 ml) and heated to 100° C. and dissolved. Aftercooling the solution to room temperature, 5.2 g (0.0375 mol) ofpotassium carbonate and 5.6 g (0.0375 mol) of sodium iodide and 6.7 ml(0.0375 mol) of benzhydrylchloride were added thereto, and the mixturewas reacted at room temperature for 18 hours under stirring. To thereaction solution, ice water was gradually added, and formedprecipitates were collected by filtration, washed sequentially withwater and ethyl ether/n-hexane (2/1), and dried to obtain 12.3 g (yield:100%) of the above identified compound.

NMR (CDCl₃)δ(ppm): 4.35(2,s), 5.96(1H,s), 6.06(1H,s), 6.55(1H,s),6.75(1H,s), 7.26(20H,s).

(4) 2-Phthalimidooxymethyl-1,5-dibenzhydryloxy-4-pyridone ##STR19##

12.3 g (0.0251 mol) of the product of Step (3) was added and dissolvedin dimethylformamide (125 ml). Then, dried tetrahydrofuran (250 ml), 4.1g (0.0251 mol) of N-hydroxyphthalimide and 9.9 g (0.0376 mol) oftriphenylphosphine were added thereto. Further, 5.8 ml (0.0377 mol) ofdiethyl diazodicarboxylate was dropwise added under cooling with ice.The mixture was stirred at the same temperature for 10 minutes, and thenethyl acetate (500 ml) and water (1,500 ml) were added thereto. Theethyl acetate layer was washed with water and a saturated sodiumchloride solution, and then dried over anhydrous magnesium sulfate, andthen concentrated to dryness. The residue was subjected to silica gelcolumn chromatography and eluted by benzene/ethyl acetate (4/1) toobtain 8.0 g of the above identified compound (yield: 50.2%)

NMR(CDCl₃)δ(ppm): 4.97(2H,s), 5.88(1H,s), 6.26(1H,s), 6.73(1H,s)6.84(1H,s), 7.31(10H,s), 7.44(10H,s), 7.76(4H,s).

(5)(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)aceticacid ##STR20## 8.0 g (0.0126 mol) of the product of Step (4) wassuspended in ethanol (60 ml). Then, 0.629 g (0.0126 mol) of hydrazinemonohydrate was added thereto, and the mixture was refluxed for 1 hour.After cooling the reaction solution to room temperature, insolubles wereremoved by filtration, and the filtrate was concentrated to dryness. Theresidue was suspended in chloroform (120 ml), and insolubles wereremoved by filtration. The filtrate was concentrated to dryness and thendissolved in ethanol (60 ml). Then, a chloroform (180 ml) solution of5.2 g (0.0126 mol) of 2-tritylaminothiazol-4-ylglyoxylic acid was addedthereto. This solution was reacted at room temperature for 18 hoursunder stirring, and then concentrated to dryness. Ethanol and n-hexanewere added thereto, and formed precipitates were collected by filtrationto obtain 9.5 g (yield: 83.8%) of the above identified compound.

NMR(CDCl₃)6(ppm):

5.02(2H,s), 5.86(1H,s), 6.24(1H,s), 6.53(1H,s),

6.74(1H,s), 6.98(1H,s), 6.89-7.50(35H,bs).

EXAMPLE 2

p-Methoxybenzyl (6R,7R)-7-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-chloromethyl-3-cephem-4-carboxylate##STR21## 9.38 g (0.01 mol) of p-methoxybenzyl7-amino-3-chloromethyl-3-cephem-4-carboxylate p-toluene sulfonate wasadded to a solvent mixture of ethyl acetate (300 ml) and water (100 ml),and 2.18 g (0.026 mol) of sodium hydorgencarbonate was added theretounder cooling with ice. The mixture was stirred for 1 hour. The ethylacetate layer was washed with a saturated sodium chloride aqueoussolution, and dried over anhydrous magnesium sulfate. To this solution,a chloroform solution (500 ml) of 6.25 g (0.00693 mol) of(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)aceticacid obtained in Example 1, 1.0 g (0.00658 mol) of1-hydroxybenzotriazole and 1.66 g (0.00624 mol) ofdicyclohexylcarbodiimide were added under cooling with ice, and themixture was reacted for 18 hours under stirring. Precipitated insolubleswere removed by filtration, and the filtrate was concentrated todryness. Ethyl acetate (300 ml) was added thereto, and insolubles wereremoved by filtration. The filtrate was concentrated to dryness, andsubjected to silica gel column chromatography and eluted withbenzene/ethyl acetate (5/1) to obtain 5.45 g (yield: 62.8%) of the aboveidentified compound.

NMR(CDCl₃)δ(ppm):

3.42(2H,bs), 3.75(3H,s), 4.32,4.69(2H,ABq,J=12 Hz), 4.89(1H,d,J=6 Hz),4.94(2H,s), 5.21(2H,s), 5.74(1H,dd,J=9 Hz, J=7 Hz), 6.05(1H,s),6.11(1H,s), 6.43(1H,s), 6.71(1H,s), 6.77(1H,s), 7.31(35H,s).

IR(KBr):vc=o 1880 cm¹.

EXAMPLE 3 (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3(1,2,3-thiadiazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid ##STR22##

1.242 g (1 mmol) of p-methoxybenzyl (6R,7R)-7-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-chloromethyl-3-cephem-4-carboxylateobtained in Example 2 was dissolved in a solvent mixture of 5 ml ofdimethylformamide and 20 ml of ethanol, and cooled with ice. Then, 0.21g (1.5 mmol) of sodium 1,2,3-thiadiazole-5-thiolate was added to thereaction solution, and the mixture was reacted for 2.5 hours understirring. The reaction solution was poured into ice, and precipitatedcrystals were collected by filtration and recrystallized from ethylacetate/diethyl ether to obtain 1.1 g of p-methoxybenzyl (6R,7R)-7-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)acetamide]-3(1,2,3-thiadiazol-5-ylthiomethyl)-3-cephem-4-carboxylate.

1.1 g of the carboxylate thus obtained was added to a mixture of 1.5 mlof anisole and 15 ml of trifluoroacetic acid, and reacted for 2 hoursunder stirring. The reaction solution was concentrated, and 100 ml ofdiethyl ether was added thereto. Formed precipitates were collected byfiltration to obtain 0.67 g of a trifluoroacetate of the aboveidentified compound.

NMR(DMSO-d₆)δ(ppm): 3.60(2H,bs), 4.30(2H,bs), 5.30(3H,m), 5.75(1H,bs),6.95(1H,s), 7.10(1H,s), 8.15(1H,s), 8.75(1H,s).

IR(KBr):vc=0, 1790cm¹.

0.67 g of the above trifluoroacetate was suspended in 20 ml of water,and adjusted to pH 7.0 with a 2% sodium hydrogencarbonate solution. Thesolution thus obtained was subjected to column chromatography withAmberlite XAD-2 (manufactured by Rohm & Haas Co.). The fraction elutedwith methanol/water (4/1) was concentrated and freeze-dried to obtain0.482 g of a sodium salt of the above identified compound.

EXAMPLE 4 (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3(2-carboxy-5-methyl-s-triazolo[1,5-a]pyrimidin-7-ylthiomethyl)-3-cephem-4-carboxylicacid ##STR23##

4.465 g (5 mmol) of(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)aceticacid obtained in Example 1 was dissolved in 50 ml of dimethylacetamide.Then, 0.766 g (5 mmol) of 1-hydroxybenzotriazole and 1.03 g (5 mmol) ofdicyclohexyl carbodiimide were added thereto under cooling with ice, andthe mixture was stirred on ice bath for 1 hour. Then, 3.82 g (6 mmol) ofp-methoxybenzyl7-amino-3-(2-benzhydryloxycarbonyl-5-methyl-s-triazolo[1,5-a]pyrimidin-7-ylthiomethyl)-3-cephem-4-carboxylatewas added thereto, and the mixture was reacted at room temperature for16 hours under stirring. Precipitated insolubles were removed byfiltration, and the filtrate was concentrated to dryness and subjectedto silica gel column chromatography and eluted with benzene/ethylacetate (3/1 to 1/2) to obtain 3.8 g of p-methoxybenzyl (6R,7R)-7-[(Z)-2-(2-tritylaminothiazol-4-yl-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(benzhydryloxycarbonyl-5-methyl-s-triazolo-[1,5-a]-pyrimidin-7-ylthiomethyl)-3-cephem-4-carboxylate.

2.4 g of the carboxylate thus obtained was dissolved in a mixture of 2ml of anisole and 9 ml of trifluoroacetic acid, and reacted at roomtemperature for 2 hours. The reaction solution was poured into 150 ml ofdiethyl ether, and formed precipitates were collected by filtration toobtain 1.252 g of a trifluoroacetate of the above identified compound.

NMR(DMSO-d₆)δ(ppm):

2.31(3H,s), 3.69(2H,bs), 5.03(1H,bs), 5.13(2H,s), 5.94(1H,bs),6.91(1H,s), 7.11(1H,s), 7.38(1H,s), 8.23(1H,s).

IR(KBr):vc=o 1780 cm⁻¹.

1.12 g of the above trifluoroacetate was suspended in 50 ml of water andadjusted to pH 7 with a sodium hydrogencarbonate solution. The solutionthus obtained was washed with ethyl acetate, and the aqueous phase wassubjected to column chromatography with HP-20 (manufactured byMitsubishi Chemical Industries Limited). The fraction eluted withethanol/water (4/1) was concentrated and freeze-dried to obtain 0.5 g ofa sodium salt of the above identified compound.

EXAMPLE 5

(6R, 7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-

dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3(1-methyl-1H-tetrazol-5-ylthiomethyl)-3-cephem-4carboxylicacid ##STR24##

4.465 g (5 mmol) of(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)aceticacid obtained in Example 1 was dissolved in 50 ml of dichloromethane.Then, 0.766 g (5 mmol) of 1-hydroxybenzotriazole and 1.03 g (5 mmol) ofdicyclohexylcarbodiimide were added under cooling with ice, and themixture was stirred for 1 hour on ice bath. Then, 2.473 g (5 mmol) ofbenzhydryl7-amino-3-(1-methyl-1H-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylatewas added thereto, and the mixture was stirred at room temperature for16 hours under stirring. Precipitated insolubles were removed byfiltration, and the filtrate was concentrated to dryness and subjectedto silica gel column chromatography and eluted with benzene/ethylacetate (4/1 to 1/2) to obtain 4.6 g of benzhydryl (6R,7R)-7-[(Z)-2(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(1-methyl-1H-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylate.

4.1 g of the carboxylate thus prepared was dissolved in a mixture of 2ml of anisole and 12 ml of trifluoroacetic acid, and the mixture wasreacted at room temperature for 2 hours. The reaction solution waspoured into 50 ml of diethyl ether, and formed precipitates werecollected by filtration to obtain 1.968 g of a trifluoroacetate of theabove identified compound.

NMR(DMSO-d₆)δ(ppm): 3.69(2H,bs), 3.95(3H,s), 4.35(2H,bs),5.19(1H,d,J=4Hz), 5.39(2H,s), 5.82(1H,bs), 6.96(1H,s), 7.19(1H,s),8.25(1H,s), 9.80(1H,d,J=8Hz).

KR(KBr):vc=o 1790 cm⁻¹.

1.268 g of the above trifluoroacetate was suspended in 30 ml of waterand adjusted to pH 7 with a sodium hydrogencarbonate solution. Thesolution thus obtained was washed with ethyl acetate, and the aqueouslayer was subjected to HP-20 column chromatography. The fraction elutedwith ethanol/water (4/1) was concentrated and freeze-dried to obtain 0.3g of a sodium salt of the above identified compound.

EXAMPLE 6 (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3[-(1(2-hydroxyethyl)-1H-tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid ##STR25##

1.242 g (1 mmol) of p-methoxybenzyl (6R,7R)-7-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-chloromethyl-3-cephem-4-carboxylateobtained in Example 2 was dissolved in a solvent mixture of 10 ml ofdimethylformamide and 20 ml of ethanol. Then, 0.161 g (1.1 mmol) of1-(2-hydroxyethyl)-1H-tetrazole-5-thiol in 35 ml of ethanol and 0.21 ml(1.1 mmol) of 5.2N sodium methoxide were added thereto under coolingwith ice, and the mixture was reacted for 25 hours under stirring. Thereaction solution was poured into ice. Precipitated crystals werecollected by filtration and subjected to silica gel columnchromatography and eluted with chloroform/methanol (30/1) to obtain 0.97g of p-methoxybenzyl(6R,7R)-7-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)acetamido]-3[-1-(2-hydroxyethyl)-1H-tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylate.

0.97 g of the carboxylate thus prepared was added to a mixture of 10 mlof trifluoroacetic acid and 1 ml of anisole, and reacted for 1 hourunder stirring and cooling with water. The reaction solution wasconcentrated, and 100 ml of diethyl ether was added thereto. Formedprecipitates were collected by filtration to obtain 0.535 g of atrifluoroacetate of the above identified compound.

NMR(DMSO-d₆) δ(ppm):

3.75(4H,bs), 4.35(4H,bs), 6.20(1H,bs), 6.40(2H,bs),

6.80(1H,bs), 7.00(1H,s), 7.15(1H,s), 8.17(1H,s),

9,80(1H,d).

IR(KBr): νc=o 1790 cm⁻¹.

0.535 g of the above trifluoroacetate was suspended in 20 ml of water,and adjusted to pH 7.0 with a 2% sodium hydrogencarbonate solution. Thesolution thus obtained was subjected to Amberlite XAD-2 columnchromatography to obtain 0.4 g of a sodium salt of the above identifiedcompound.

EXAMPLE 7 (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(1-carboxymethyl-1H-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid ##STR26##

7.145 g (8 mmol) of (Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)-acetic acid obtainedin Example 1 was dissolved in 130 ml of dichloromethane. Then, 1.225 g(8 mmol) of 1-hydroxybenzotriazole and 1.648 g (8 mmol) of dicyclohexylcarbodiimide were added thereto under cooling with ice, and the mixturewas stirred for 1 hour on ice bath. Then, 6.195 g (8.8 mmol) ofbenzhydryl7-amino-3-(1-benzhydryloxycarbonylmethyl-1H-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylatewas added thereto, and reacted at room temperature for 16 hours understirring. Precipitated insolubles were removed by filtration, and thefiltrate was concentrated to dryness and subjected to silica gel columnchromatography and eluted with chloroform/acetone (50/1 to 10/1) toobtain 11.4 g of benzhydryl (6R,7R)-7-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(1-benzhydryloxycarbonylmethyl-1H-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylate.

10 g of the carboxylate thus obtained was dissolved in a mixture of 5 mlof anisole and 50 ml of trifluoroacetic acid, and reacted at roomtemperature for 2 hours. The reaction solution was poured into 400 ml ofdiethyl ether, and formed precipitates were collected by filtration toobtain 5.0 g of a trifluoroacetate of the above identified compound.

NMR(DMSO-d₆) δ(ppm):

3.73(2H,bs), 4.40(2H,bs), 5,32(5H,s), 5.83(1H,bs),

6.97(1H,s), 7.20(1H,s), 8.27(1H,s), 9.85(1H,bs).

IR(KBr) νc=o: 1780 cm⁻¹.

5.0 g of the above trifluoroacetate was suspended in 100 ml of water,and adjusted to pH 7 with a sodium hydrogencarbonate solution. Thesolution thus obtained was washed with ethyl acetate, and the aqueousphase was subjected to HP-20 column chromatography. The fraction elutedwith ethanol/water (4/1) was concentrated and freeze-dried to obtain0.85 g of a sodium salt of the above identified compound.

EXAMPLE 8

(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-[1-(2-dimethylaminoethyl)-1H-tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid ##STR27##

1.242 g (1 mmol) of p-methoxybenzyl (6R,7R)-7-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-chloromethyl-3-cephem-4-carboxylateobtained in Example 2 was dissolved in 10 ml of dimethylformamide. Then,0.213 g (1.1 mmol) of sodium1-(2-dimethylaminoethyl)-1H-tetrazole-5-thiolate in dimethylformamidewas added thereto under cooling with ice at -20° C., and the mixture wasreacted under stirring for 3 hour. The reaction solution was poured toice. Precipitated crystals were collected by filtration and thensubjected to silica gel column chromatography and eluted withchloroform/methanol (10/1) to obtain 0.745 g of p-methoxybenzyl (6R,7R)-7-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-[1-(2-dimethylaminoethyl)-1H-tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylate.

0.745 g of the carboxylate thus obtained was dissolved in a mixture of10 ml of trifluoroacetic acid and 1 ml of anisole, and reacted for 2hours under stirring. The reaction solution was concentrated, and then100 ml of diethyl ether was added thereto. Formed precipitates werecollected by filtration to obtain 0.490 g of a trifluoroacetate of theabove identified compound.

NMR(DMSO-d₆) δ(ppm);

2.90(6H,s), 3.70(4H,m), 4.35(2H,bs), 4.55(2H,bs),

5.20(1H,m), 5.40(2H,bs), 5.85(1H,m), 6.85(1H,s),

7.15(1H,s).

0.490 g of the above trifluoroacetate was suspended in 20 ml of water,and adjusted to pH 7.0 with a 2% sodium hydrogencarbonate solution. Thesolution thus obtained was subjected to Amberlite XAD-2 columnchromatography. The fraction eluted with ethanol/water (4/1) wasconcentrated and freeze-dried to obtain 0.294 g of a sodium salt of theabove identified compound.

IR(KBr): νc=o 1790 cm⁻¹.

The sodium salt was dissolved in water and adjusted to pH 1.1 with 1Nhydrochloric acid to obtain a hydrochloride of the above identifiedcompound.

EXAMPLE 9 (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(1,3,4-thiadiazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid ##STR28##

4.465 g (5 mmol) of(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyrion-2-ylmethoxyimino)aceticacid obtained in Example 1 was dissolved in 50 ml of dichloromethane.Then, 0.766 g (5 mmol) of 1-hydroxybenzotriazole and 1.03 g (5 mmol) ofdicyclohexylcarbodiimide were added thereto under cooling with ice, andthe mixture was stirred for 1 hour on ice bath. Then, 2.7 g (6 mmol) ofp-methoxybenzyl7-amino-3-(1,3,4-thiadiazol-5-ylthiomethyl)-3-cephem-4-carboxylate wasadded thereto, and reacted at room temperature for 16 hours understirring. Precipitated insolubles were removed by filtration, and thefiltrate was concentrated to dryness and subjected to silica gel, columnchromatography and eluted with chloroform/acetone (17/1 to 3/1) toobtain 4.1 g of p-methoxybenzyl (6R,7R)-7-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(1,3,4-thiadiazol-5-ylthiomethyl)-3-cephem-4-carboxylate.

2.1 g of the carboxylate thus obtained was dissolved in a mixture of 1ml of anisole and 6 ml of trifluoroacetic acid, and reacted at roomtemperature for 2 hours. The reaction solution was poured into 50 ml ofdiethyl ether. Formed precipitates were collected by filtration toobtain 1.254 g of a trifluoroacetate of the above identified compound.

NMR(DMSO-d₆) δ(ppm):

3.80(2H,s), 4,39(2H,bs), 5.18(1H,d,J=4Hz),

5.38(2H,s), 5.85(1H,bs), 6.96(1H,s), 7.21(1H,s),

7.34(1H,s), 8.27(1H,s).

IR(KBr): νc=o 1780 cm⁻¹.

1.0 g of the above trifluoroacetate was suspended in 50 ml of water, andadjusted to pH 7 with a sodium hydrogencarbonate solution. The solutionthus obtained was washed with ethyl acetate, and the aqueous layer wassubjected to HP-20 column chromatography. The fraction eluted withethanol/water (4/1) was concentrated and freeze-dried to obtain 0.55 gof a sodium salt of the above identified compound.

EXAMPLE 10 (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyamino)acetamido]-3-(2-methyl-1,3,4-thiadiazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid ##STR29##

3.57 g (4 mmol) of(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)aceticacid obtained in Example 1 was dissolved in 80 ml of dichloromethane.Then, 0.613 g (4 mmol) of 1-hydroxybenzotriazole and 0.824 g (4 mmol) ofdicyclohexylcarbodiimide were added thereto under cooling with ice, andthe mixture was stirred for 1 hour on ice bath. Then, 1.86 g (4 mmol) ofp-methoxybenzyl7-amino-3-(2-methyl-1,3,4-thiadiazol-5-ylthiomethyl)-3-cephem-4-carboxylatewas added thereto, and reacted at room temperature for 18 hours understirring. Precipitated insolubles were removed by filtration, and thefiltrate was concentrated to dryness, and subjected to silica gel columnchromatography and eluted with chloroform/ethyl acetate (1/3) to obtain1.3 g of p-methoxybenzyl (6R,7R)-7-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)-acetamido]-3-(2-methyl-1,3,4-thiadiazol-5-ylthiomethyl)-3-cephem-4-carboxylate.1.0 g of the carboxylate thus prepared was dissolved in a mixture of 1ml of anisole and 3.3 ml of trifluoroacetic acid, and reacted at roomtemperature for 3 hours. The reaction solution was poured into 30 ml ofdiethyl ether, and formed precipitates were collected by filtration toobtain 0.6 g of a trifluoroacetate of the above identified compound.

NMR(DMSO-d₆) δ(ppm):

2.67(3H,s), 3.73(2H,s), 4.20(2H,bs), 5.14(1H,bs),

5.36(2H,s), 5.80(1H,bs), 6.90(1H,s), 7.24(1H,s),

8.20(1H,s).

IR(KBr): νc=o 1770 cm⁻¹.

0.6 g of the above trifluoroacetate was suspended in 10 ml of water, andadjusted to pH 7 with a sodium hydrogencarbonate solution. The solutionthus obtained was washed with ethyl acetate, and the aqueous phase wassubjected to XAD-2 column chromatography. The fraction eluted withmethanol/water (4/1) was concentrated and freeze-dried to obtain 0.314 gof a sodium salt of the above identified compound.

EXAMPLE 11 (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(1,2,3-triazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid ##STR30##

5.4 g (6 mmol) of(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)aceticacid obtained in Example 1 was dissolved in 100 ml of dichloromethane.Then, 0.918 g (6 mmol) of 1-hydroxybenzotriazole and 1.24 g (6 mmol) ofdicyclohexyl carbodiimide were added thereto under cooling with ice, andthe mixture was stirred for 1 hour on ice bath. Then, 3.5 g (6.7 mmol)of p-methoxybenzyl7-amino-3-(1-tetrahydropyranyl-1,2,3-triazol-5-ylthiomethyl)-3-cephem-4-carboxylatewas added thereto, and reacted at room temperature for 18 hours understirring. Precipitated insolubles were removed by filtration, and thefiltrate was concentrated by dryness and subjected to silica gel columnchromatography and eluted with chloroform/acetone (2/1) to obtain 4.6 gof p-methoxybenzyl (6R,7R)-7-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(1-tetrahydropyranyl-1,2,3-triazol-5-ylthiomethyl)-3-cephem-4-carboxylate.

1.0 g of the carboxylate thus obtained was dissolved in a mixture of 1ml of anisole and 3.3 ml of trifluoroacetic acid, and reacted at roomtemperature for hours. The reaction solution was poured into 30 ml ofdiethyl ether, and formed precipitates were collected by filtration toobtain 0.48 g of a trifluoroacetate of the above identified compound.

NMR(DMSO-d₆) δ(ppm)

3.70(2H,bs), 4.00(2H,bs), 5.12(1H,bs), 5.20(2H,s),

5.73(1H,bs), 6.82(1H,s), 6.88(1H,s), 7.27(1H,s), 7.90(1H,s).

KR(KBr: νc=o 1775 cm⁻¹.

0.48 g of the above trifluoroacetate was suspended in 50 ml of water,and adjusted to pH 7 with a sodium hydrogencarbonate solution. Thesolution thus obtained was washed with ethyl acetate, and the aqueousphase was subjected to XAD-2 column chromatography. The fraction elutedwith methanol/water (4/1) was concentrated and freeze-dried to obtain0.15 g of a sodium salt of the above identified compound.

EXAMPLE 12 (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(2,5-dihydro-6-hydroxy-2-methyl-5-oxo-as-triazin-3-ylthiomethyl)-3-cephem-4-carboxylicacid ##STR31##

7.415 g (8 mmol) of(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)-aceticacid obtained in Example 1 was dissolved in 50 ml of dichloromethane.Then, 1.225 g (8 mmol) of 1-hydroxybenzotriazole and 1.648 g (8 mmol) ofdicyclohexylcarbodiimide were added thereto under cooling with ice, andthe mixture was stirred for 1 hour on ice bath. Then, 6.186 g (8.8 mmol)of benzhydryl7-amino-3-(2,5-dihydro-6-benzhydryloxy-2-methyl-5-oxo-astriazin-3-ylthiomethyl)-3-cephem-4-carboxylatewas added thereto, and reacted at room temperature for 16 hours understirring. Precipitated insolubles were removed by filtration, and thefiltrate was concentrated to dryness, and subjected to silica gel columnchromatography and eluted with chloroform/acetone (50/1 to 10/1) toobtain 11 g of benzhydryl (6R,7R)-7-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)-acetamido]-3-(2,5-dihydro-6-benzhydryloxy-2-methyl-5-oxo-as-triazin-3-ylthiomethyl)-3-cephem-4-carboxylate.

11 g of the carboxylate thus obtained was dissolved in a mixture of 5 mlof anisole and 50 ml of trifluoroacetic acid, and the mixture wasreacted at room temperature for 2 hours. The reaction solution waspoured into 400 ml of diethyl ether. Formed precipitates were collectedby filtration to obtain 7.0 g of a trifluoroacetate of the aboveidentified compound.

NMR(DMSO-d₆) δ(ppm):

3.67(2H,s), 3.76(3H,s), 4.24(2H,bs),

5.23-5.94(4H,bs), 7.04(1H,s), 7.28(1H,s),

8.32(1H,s), 9.93(1Hbs).

IR(KBr): νc=o 1780 cm⁻¹.

4.572 g of the above trifluoroacetate was suspended in 100 ml of water,and adjusted to pH 7 with a sodium hydrogencarbonate solution. Thesolution thus obtained was washed with ethyl acetate, and the aqueousphase was subjected to HP-20 column chromatography. The fraction elutedwith ethanol/water (4/1) was concentrated and freeze-dried to obtain1.89 g of a sodium salt of the above identified compound.

EXAMPLE 13 (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(5-ethoxycarbonylmethyl-4-methylthiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid ##STR32##

4.465 g (5 mmol) of(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)aceticacid obtained in Example 1 was dissolved in 50 ml of dichloromethane.Then, 0.766 g (5 mmol) of 1-hydroxybenzotriazole and 1.03 g (5 mmol) ofdicyclohexyl carbodiimide were added thereto under cooling with ice, andthe mixture was stirred for 1 hour on ice bath. Then, 4.146 g (6 mmol)of p-methoxybenzyl7-amino-3-(5-ethoxycarbonylmethyl-4-methylthiazol-2-ylthiomethyl)-3-cephem-4-carboxylatewas added, and reacted at room temperature for 16 hour under stirring.Precipitated insolubles were removed by filtration, and the filtrate wasconcentrated to dryness, and subjected to silica gel columnchromatography and eluted with chloroform/ethyl acetate (9/1 to 1/2) toobtain 5.0 g of p-methoxybenzyl (6R,7R)-7-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(5-ethoxycarbonylmethyl-4-methylthiazol-2-ylthiomethyl)-3-cephem-4-carboxylate.

0.5 g of the carboxylate thus obtained was dissolved in a mixture of 2ml of anisole and 12 ml of trifluoroacetic acid, and the mixture wasreacted at room temperature for 2 hours. The reaction solution waspoured into 160 ml of diethyl ether, and formed precipitates werecollected by filtration to obtain 2.798 g of a trifluoroacetate of theabove identified compound.

NMR(DMSO-d₆) δ(ppm): 1.20(3H,t,J=7Hz), 2.26(3H,s), 3.72(2H,bs),3.82(2H,s), 3.86-4.35(4H,m), 5.15(1H,d,J=4Hz), 5.38(2H,s), 6.17(1H,bs),6.91(1H,s), 7.16(1H,s), 8.23(1H,s), 9.80(1H,d,J=7Hz).

IR(KBr): νc=o 1790 cm³¹ 1.

2.5 g of the above-mentioned trifluoroacetate was suspended in 100 ml ofwater, and adjusted to pH 7 with a sodium hydrogencarbonate solution.The solution thus obtained was washed with ethyl acetate, and theaqueous phase was subjected to HP-20 column chromatography. The fractioneluted with ethanol/water (4/1) was concentrated and freeze-dried toobtain 1.3 g of a sodium salt of the above identified compound.

EXAMPLE 14

(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(4-carboxy-3-hydroxyisothiazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid ##STR33##

150 mg (0.116 mmol) of benzhydryl (6R,7R)-7-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-chloromethyl-3-cephem-4-carboxylateobtained in Example 2 was added to a solvent mixture of 4.5 ml ofdimethylformamide, 0.5 ml of methanol and 0.2 ml of water. Then, 30 mg(0.123 mmol) of trisodium 4-carboxy-3-hydroxy-5-mercaptoisothiazole wasadded thereto, and reacted at room temperature for 18 hours. Thereaction solution was added to a solvent mixture of 30 ml ofdichloromethane and 20 ml of dilute hydrochloric acid, and thedichloromethane layer was washed with water and dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and ethyl ether was added to the residue, followed by conversion intopowder to obtain 85 g of benzhydryl (6R,7R)-7-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)-acetamido]-3-(4-carboxy-3-hydroxyisothiazol-5-ylthiomethyl)-3-cephem-4-carboxylate.

80 mg of the carboxylate thus obtained was added to a mixture of 0.07 mlof anisole and 0.9 ml of trifluoroacetic acid, and the mixture wasreacted at room temperature for 3 hours under stirring. The solvent wasdistilled off under reduced pressure, and ethyl ether was added to theresidue. Formed precipitates were collected by filtration to obtain 40mg of a trifluoroacetate of the above identified compound.

NMR(DMSO-d₆) δ(ppm):

3.47(2H,bs), 3.67(2H,s), 5.36(3H,bs), 5.90(1H,m)

6.94(1H,s), 7.16(1H,s), 8.25(1H,s).

IR(KBr): νc=o 1770 cm⁻¹.

40 mg of the above trifluoroacetate was added to 10 ml of water, andadjusted to pH 7.0 with a 2% sodium hydrogencarbonate solution. Thesolution thus obtained was subjected to XAD-2 column chromatography. Thefraction eluted with ethanol/water (4/1) was concentrated andfreeze-dried to obtain 30 mg of a sodium salt of the above identifiedcompound.

EXAMPLE 15 (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-acetoxymethyl-3-cephem-4-carboxylicacid ##STR34##

1.786 g (2 mmol) of(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)aceticacid obtained in Example 1 was dissolved in 20 ml of dichloromethane.Then, 0.308 g (2 mmol) of 1-hydroxy-benzotriazole and 0.412 g (2 mmol)of dicyclohexyl carbodiimide were added thereto under cooling with ice,and the mixture was stirred for 1 hour on ice bath. Then, 0.691 g (2mmol) of t-butyl 7-amino-3-acetoxymethyl-3-cephem-4-carboxylate wasadded thereto, and reacted at room temperature for 16 hours understirring. Precipitated insolubles were removed by filtration, and thefiltrate was concentrated to dryness and subjected to silica gel columnchromatography and eluted with benzene/ethyl acetate (2/1 to 1/2) toobtain 1.1 g of t-butyl (6R,7R)-7-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-acetoxymethyl-3-cephem-4-carboxylate.

1.1 g of the carboxylate thus prepared was dissolved in a mixture of 2ml of anisole and 8 ml of trifluoroacetic acid, and the mixture wasreacted at room temperature for 2 hours. The reaction solution waspoured into 50 ml of diethyl ether, and formed precipitates werecollected by filtration to obtain 0.577 g of a trifluoroacetate of theabove identified compound.

NMR(DMSO-d₆) δ(ppm):

2.07(3H,s), 3.58(2H,s), 5.25(1H,d,J=4Hz),

5.45(2H,s), 5.92(1H,bs), 7.03(1H,s),7.27(1H,s) 8.14(1H,s), 9.88(1H,bs).

IR(KBr): νc=o 1780 cm⁻¹.

0 5 g of the above trifluoroacetate was suspended in 10 ml of water andadjusted to pH 7 with a sodium hydrogencarbonate solution. The solutionthus obtained was washed with ethyl acetae, and the aqueous phase wassubjected to HP-20 column chromatography. The fraction eluted withethanol/water (4/1) was concentrated and freeze-dried to obtain 0.25 gof a sodium salt of the above identified compound.

EXAMPLE 16 (6R,7R)-7-[(Z)-2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-vinyl-3-cephem-4-carboxylicacid ##STR35##

1.85 g (2 mmol) of(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)-aceticacid obtained in Example 1 was dissolved in 20 ml of dimethylacetamido.Then, 0.315 g (2 mmol) of 1-hydroxybenzotriazole and 0.423 g (2 mmol) ofdicyclohexylcarbodiimide were added thereto under cooling with ice, andthe mixture was stirred for 1 hour on ice bath. Then, 5 ml of adimethylacetamide solution of 0.8 g (2.33 mmol) of p-methoxybenzyl7-amino-3-vinyl-3-cephem-4-carboxylate was added thereto, and reacted atroom temperature for 18 hours under stirring. Precipitated insolubleswere removed by filtration, and the filtrate was concentrated todryness, and subjected to silica gel column chromatography and elutedwith benzene/ethyl acetate (1/3) to obtain 1.3 g of p-methoxybenzyl (6R,7R)-7-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-vinyl-3-cephem-4-carboxylate.

1.2 g of the carboxylate thus prepared was dissolved in a mixture of 1.3ml of anisole and 4.4 ml of trifluoroacetic acid, and the mixture wasreacted at room temperature for 3 hours. The reaction solution waspoured into 30 ml of diethyl ether, and formed precipitates werecollected by filtration to obtain a trifluoroacetic acid of the aboveidentified compound.

NMR(CD₃ COCD₃) δ(ppm):

3.72(2H,bs), 5.28(1H,d), 5.45(2H,s), 5.93(1H,bs)

7.05(1H,s), 7.22(1H,s), 8.22(1H,s).

IR(KBr): νc=o 1770 cm⁻¹.

0.6 g of the above trifluoroacetate was suspended in 30 ml of water, andadjusted to pH 7 with sodium hydrogencarbonate solution. This solutionwas washed with ethyl acetate, and the aqueous layer was subjected toAmberlite XAD-2 column chromatography. The fraction eluted withmethanol/water (4/1) was concentrated and freeze-dried to obtain 0.2 gof a sodium salt of the above identified compound.

EXAMPLE 17 (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(5-methyl-2H-tetrazol-2-ylmethyl)-3-cephem-4-carboxylicacid ##STR36##

6.31 g (7 mmol) of(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)aceticacid was dissolved in 70 ml of tetrahydrofuran. Then, 1.07 g (7 mmol) of1-hydroxybenzotriazole and 1.59 g (7.7 mmol) of dicyclohexylcarbodiimide were added thereto under cooling with ice, and the mixturewas stirred for 1 hour in ice bath. Then, a solution obtained bydissolving 3.24 g (7 mmol) of p-methoxybenzyl7-amino-3-(5-methyl-2H-tetrazol-2-ylmethyl)-3-cephem-4-carboxylate in 70ml of tetrahydrofuran, was added thereto, and the mixture was reacted atroom temperature for 16 hours under stirring. Precipitated insolubleswere removed by filtration, and the filtrate was concentrated to drynessand subjected to silica gel column chromatography and eluted withchloroform/acetone (100/1 to 10/1) to obtain 6.32 g of p-methoxybenzyl(6R,7R)-7-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-benzhydryloxy-4-pyridon-2-ylmethoxyimino)-acetamido]-3-(5-methyl-2H-tetrazol-2-ylmethyl)-3-cephem-4-carboxylate.

6.01 g of the carboxylate thus obtained was dissolved in a mixture of 6ml of anisole and 30 ml of trifluoroacetic acid, and the mixture wasreacted at room temperature for 1.5 hours. The reaction solution waspoured into 200 ml of diisopropyl ether, and formed precipitates werecollected by filtration to obtain 3.57 g of a trifluoroacetate of theabove identified compound.

NMR(DMSO-d₆) δ(ppm):

2.48(3H,s), 3.45(2H,bs), 5.23(1H,d,J=4Hz),

5.38(2H,bs), 5.70(2H,bs), 5.92(1H,dd,J=4.8Hz),

6.97(1H,s), 7.18(1H,s), 8.28(1H,s).

IR(KBr): νc=o 1790 cm⁻¹.

3.5 g of the above trifluoroacetate was suspended in 30 ml of water, and960 mg of sodium hydrogencarbonate was added to obtain a uniformsolution. The solution thus obtained was washed with ethyl acetate, andthe aqueous phase was subjected to HP-20 column chromatography. Thefraction eluted with ethanol/water (4/1) was concentrated andfreeze-dried to obtain 2.1 g of a sodium salt of the above identifiedcompound.

EXAMPLE 18 (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(1-methylpyridinium-4-ylthiomethyl)-3-cephem-4-carboxylat##STR37##

4.3 g (3.43 mmol) of the product of Example 2 was dissolved in drytetrahydrofuran (200 ml), and 1.17 g (10.3 mmol) of1-methylpyridine-4-thione was added thereto. The mixture was reacted for18 hours under stirring and cooling with ice, the reaction solution wasconcentrated under reduced pressure, and then subjected to silica gelcolumn chromatography and eluted with chloroform/methanol (10/1) toobtain 2.8 g (yield: 59.7%) of p-methoxybenzyl(6R,7R)-7-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(1-methylpyridinium-4-ylthiomethyl)-3-cephem-4-carboxylatechloride. This product was dissolved in a mixture of trifluoroaceticacid (14 ml) and anisole (2.8 ml). The mixture was reacted for 1 hourunder stirring and cooling with ice. Ethyl ether (140 ml) was added tothe reaction solution, and formed precipitates were collected byfiltration to obtain a trifluoroacetate of the above identifiedcompound.

NMR(DMSO-d₆) δ(ppm):

3.68(2H,bs), 4.20(3H,s), 4.40(2H,bs), 5.36(2H,s),

6.89(1H,s), 7.16(1H,s), 7.97(2H,d,J=6.4Hz),

8.21(1H,s), 8.67(2H,d,J=6.4Hz).

IR(KBr): νc=o 1780 cm⁻¹.

The above trifluoroacetate was suspended in water (40 ml), and adjustedto pH 7.0 with a 2% sodium hydrogencarbonate solution. Then, it wassubjected to HP-20 column chromatography to obtain 0.86 g (yield: 66.7%)of the above identified compound.

EXAMPLE 19 (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(2,3-cyclopenteno-1-methylpyridinium-4-ylthiomethy)-3-cephem-4-carboxylate##STR38##

2.6 g (2.1 mmol) of the product of Example 2 was dissolved indimethylformamide (40 ml), and 1.03 g (6.3 mmol) of1-methylcyclopenteno[b]pyridine-4-thione was added thereto. The mixturewas reacted at room temperature for 3 days under stirring. Chloroform(400 ml) was added to the reaction solution, and washed sequentiallywith 0.1N hydrochloric acid and a saturated sodium chloride aqueoussolution, then dried over anhydrous magnesium sulfate and concentratedto dryness. The residue was subjected to silica gel columnchromatography and eluted with chloroform/methanol (10/1) to obtain 1.63g (yield: 55.4%) of p-methoxybenzyl (6R,7R)-7-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(2,3-cyclopenteno-1-methylpyridinium-4-ylthiomethyl)-3-cephem-4-carboxylatechloride. This was dissolved in a mixture of trifluoroacetic acid (8 ml)and anisole (1.6 ml), and the mixture was reacted for 1 hour understirring and cooling with ice. Ethyl ether (80 ml) was added to thereaction solution, and formed precipitates were collected by filtrationto obtain a trifluoroacetate of the above identified compound.

NMR(DMSO-d₆) δ(ppm):

1.85-2.51(2H,m), 2.68-3.52(4H,m), 3.70(2H,bs),

4.11(3H,s), 4.46(2H,bs), 5.38(2H,s), 6.95(1H,s),

7.29(1H,s), 7.86(1H,d,J=7Hz), 8.38(1H,s),

8.65(1H,d,J=7Hz).

IR(KBr): νc=o 1768 cm⁻¹.

The above trifluoroacetate was suspended in water (20 ml), and adjustedto pH 7.0 with a 2% sodium hydrogencarbonate solution. Then, it wassubjected to HP-20 column chromatography to obtain 0.36 g (yield: 45.6%)of the above identified compound.

EXAMPLE 20 Sodium (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(1-carboxymethylpyridinium-4-ylthiomethyl)-3-cephem-4-carboxylate##STR39##

2.6 g (2.1 mmol) of the product of Example 2 was dissolved in drytetrahydrofuran (50 ml). Then, 1.42 g (6.3 mmol) of1-t-butoxycarbonylmethylpyridine-4-thione was added thereto, and themixture was reacted at room temperature for 2 days under stirring. Thereaction solution was concentrated under reduced pressure and thensubjected to silica gel column chromatography and eluted withchloroform/methanol (10/1) to obtain 1.65 g (yield: 53.7%) ofp-methoxybenzyl (6R,7R)-7-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(1-t-butoxycarbonylmethylpyridinium-4-ylthiomethyl)-3-cephem-4-carboxylatechloride.

This product was dissolved in a mixture of trifluoroacetic acid (17 ml)and anisole (3.4 ml), and reacted at room temperature for 4 hours understirring. Ethyl ether (200 ml) was added to the reaction solution, andformed precipitates were collected by filtration to obtain atrifluoroacetate of the above identified compound.

NMR(DMSO-d⁶) δ(ppm):

3.68(2H,bs), 4.40(2H,bs), 5.36(2H,s), 5.37(2H,s),

6.90(1H,s), 7.13(1H,s), 8.06(2H,d,J=7Hz),

8.23(1H,s), 8.76(2H,d,J=7Hz).

IR(KBr): νc=o 1780 cm⁻¹.

The above trifluoroacetate was suspended in water (80 ml), and adjustedto pH 7.0 with a 2% sodium hydrogencarbonate solution. Then, it wassubjected to HP-20 column chromatography to obtain 0.321 g (yield:40.4%) of the above identified compound.

EXAMPLE 21 Sodium (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(1-carboxymethyl-2,3-cyclopentenopyridinium-4-ylthiomethyl)-3-cephem-4-carboxylate##STR40##

2.6 g (2.1 mmol) of the product of Example 2 was dissolved in drytetrahydrofuran (50 ml), and 1.67 g (6.3 mmol) of1-t-butoxycarbonylmethyl-cyclopenteno[b]pyridine- 4-thione. The mixturewas reacted at room temperature for 2 days under stirring. The reactionsolution was concentrated under reduced pressure, and subjected tosilica gel column chromatography and eluted with chloroform/methanol(10/1) to obtain 1.8 g (56.4%) of p-methoxybenzyl (6R,7R)-7-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(1-t-butoxycarbonylmethyl-2,3-cyclopentenopyridinium-4-ylthiomethyl)-3-cephem-4-carboxylatechloride. This product was dissolved in a mixture of trifluoroaceticacid (18 ml) and anisole (3.6 ml). Ethyl ether (240 ml) was added to thereaction solution, and formed precipitates were collected by filtrationto obtain a trifluoroacetate of the above identified compound.

NMR(DMSO-d₆) δ(ppm):

2.20-2.53(2H,m), 2.70-3.40(4H,m), 3.70(2H,bs),

4.44(2H,bs), 5.33(2H,bs), 5.36(2H,s), 6.93(1H,s),

7.14(1H,s), 7.92(1H,d,J=8Hz), 8.23(1H,s),

8.64(1H,d,J=8Hz).

IR(KBr): νc=o 1783 cm³¹ 1.

The above trifluoroacetate was suspended in water (80 ml), and adjustedto pH 7.0 with a 2% sodium hydrogencarbonate solution. It was thensubjected to HP-20 column chromatography, to obtain 0.517 g (yield:58.0%) of the above identified compound.

EXAMPLE 22 (6R, 7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(1-t-butoxycarbonylmethylpyridinium-4-ylthiomethyl)-3-cephem-4-carboxylate##STR41##

1.65 g (1.12 mmol) of the reaction intermediate of Example 20t-methoxybenzyl (6R,7R)-7-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(1-t-butoxycarbonylmethylpyridinium-4-ylthiomethyl)-3-cephem-4-carboxylatechloride was dissolved in a mixture of trifluoroacetic acid (8.5 ml) andanisole (1.7 mmol), and reacted for 1 hour under stirring and coolingwith ice. Ethyl ether (100 ml) was added to the reaction solution, andformed precipitates were collected by filtration to obtain atrifluoroacetate of the above identified compound.

IR(KBr): νc=o 1784 cm⁻¹.

The above trifluoroacetate was suspended in water (80 ml) and adjustedto pH 7.0 with a 2% sodium hydrogencarbonate solution. Then, it wassubjected to HP-20 column chromatography to obtain 0.325 g (yield:38.9%) of the above identified compound.

EXAMPLE 23 (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(1-t-butoxycarbonylmethyl-2,3-cyclopentenopyridinium-4-ylthiomethyl)-3-cephem-4-carboxylate##STR42##

1.8 g (1.19 mmol) of the reaction intermediate of Example 21p-methoxybenzyl (6R,7R)-7-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pypridon-2-ylmethoxyimino)acetamido]-3-(1-t-butoxycarbonylmethyl-2,3-cyclopentenopyridinium-4-ylthiomethyl)-3-cephem-4-carboxylatechloride was dissolved in a mixture of trifluoroacetic acid (9 ml) andanisole (1.8 ml), and reacted for 1 hour under stirring and cooling withice. Ethyl ether (100 ml) was added to the reaction solution, and formedprecipitates were collected by filtration to obtain a trifluoroacetateof the above identified compound.

NMR(DMSO-d₆) δ(ppm):

1.48(9H,s), 1.99-2.55(2H,m), 2.72-3.52(4H,m),

3.69(2H,bs), 4.43(2H,bs), 5.37(2H,s), 5.40(2H,s),

6.90(1H,s), 7.19(1H,s), 7.91(1H,d,J=8Hz),

8.28(1H,s), 8.65(1H,d,J=8Hz).

IR(KBr): νc=o 1782 cm⁻¹.

The above trifluoroacetate was suspended in water (80 ml), and adjustedto pH 7.0 with a 2% sodium hydrogencarbonate solution. Then, it wassubjected to HP-20 column chromatography to obtain 0.55 g (yield: 8.8%)of the above identified compound.

EXAMPLE 24 (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(5-methyl-s-triazolo[1,5-a]pyrimidin-7-ylthiomethyl)-3-cephem-4-carboxylicacid ##STR43##

3.8 g (5 mmol) of(Z)-2-(2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)aceticacid obtained in Example 1 was dissolved in 20 ml of tetrahydrofuran.Then, 765 mg (5 mmol) of 1-hydroxybenzotriazole and 1.03 g (5.5 mmol) ofcyclohexylcarbodiimide were added thereto under cooling with ice, andthe mixture was stirred for 2 hours on water bath. Then, 2.5 g (5 mmol)of p-methoxybenzyl7-amino-3-(5-methyl-s-triazolo[1,5-a]pyrimidin-7-yl-thiomethyl)-3-cephem-4-carboxylatewas added thereto, and reacted at room temperature for 16 hours understirring. Precipitated insolubles were removed by filtration, and thefiltrate was dried under reduced pressure, and then subjected to silicagel column chromatography and eluted with ether/tetrahydrofuran (2/1 to1/2) to obtain 3.0 g of p-methoxybenzyl (6R,7R)-7-[(Z)-2-tritylaminothiazol-4-yl)-2-(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxyimino)-acetamido]-3-(5-methyl-s-triazolo[1,5-a]pyrimidin-7-ylthiomethyl)-3-cephem-4-carboxylate.

This carboxylate was added to a mixture of 15 ml of trifluoroacetic acidand 3 ml of anisole, and the mixture was stirred at room temperature for2 hours. The reaction solution was poured into 200 ml of isopropylether, and formed precipitates were collected by filtration to obtain2.0 g of a trifluoroacetate of the above identified compound.

NMR(DMSO-d₆) δ(ppm):

2.60(3H,s), 3.72(2H,bs), 4.45(2H,bs),

5.22(1H,d,J=5Hz), 5.38(2H,bs), 5.85(1H,bs),

6.92(1H,s), 7.17(1H,s), 7.27(1H,s), 8.20(1H,s),

8.50(1H,s), 9.80(1H,s).

IR(KBr): νc=o 1780 cm⁻¹.

2.0 g of the above trifluoroacetate was suspended in 60 ml of water, anddissolved with an addition of 2.2 g of sodium hydrogencarbonate.Insolubles were removed by filtration, and the aqueous layer was washedwith 20 ml of n-butanol. The aqueous layer was separated.

The separated aqueous layer was adjusted to pH 3.5 by an addition of 1Nhydrochloric acid, and formed precipitates were collected by filtrationand washed sequentially with water, ethyl ether and acetone, and thenadded to a solution of 150 mg of sodium hydrogencarbonate in 15 ml ofwater, then freeze-dried to obtain 1.0 g of a sodium salt of the aboveidentified compound.

We claim:
 1. A cephalosporin compound having the formula: ##STR44##wherein R₁ is --CH₂ --A wherein A is tetrazolyl, thiazolylthio,isothiazolylthio, thiadiazxolylthio, triazolylthio, triazinylthio,tetrazxolylthio, triazolylpyrimidinylthio, pyridinium-4-ylthio or2,3-cyclopenteno-pyridinium-4ylthio group which is unsubstituted orsubstituted hby one to three groups selected from the group consistingof a C₁₋₄ alkyl group, a carboxymethyl group, a C₁₋₄alkoxycarbonylmethyl group, a hydroxy-(C₁₋₄)alkyl group, adi-(C₁₋₄)alkylamino-(C₁₋₄)alkyl group, a carboxyl group, a hydroxylgroup, an oxo group and an amino group, or a pharmaceutically acceptablesalt thereof.
 2. (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(1,2,3-thiadiazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid or a pharmaceutically acceptable salt thereof, according toclaim
 1. 3. (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-[1-(2-hydroxyethyl)-1H-tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid or a pharmaceutically acceptable salt thereof, according toclaim
 1. 4. (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(1-methylpyridinium-4-ylthiomethyl)-3-cephem-4-carboxylate,5. (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1,5-dihydroxy-4-pyridon-2-ylmethoxyimino)acetamido]-3-(5-methyl-s-triazolo[1,5-a]pyrimidin-7-ylthiomethyl)-3-cephem-4-carboxylicacid or a pharmaceutically acceptable salt thereof, according toclaim
 1. 6. An antibacterial pharmaceutical composition containing anantibacterially effective amount of a cephalosporin compound of theformula I as defined in claim 1 or a pharmaceutically acceptable saltthereof.
 7. A method of treating infectious disease which comprisesadministering an antibacterially effective amount of a cephalosporincompound of the formula I as defined in claim 1 to an animal or humanbeing.